Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. This process requires the protein to change conformations so as to expose a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, based on a diverse set of different architectures, albeit always with some form of internal structural symmetry. In spite of unprecedented insights from three-dimensional structures obtained in the last decade, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, as well as identification of the binding regions for the substrate or substrates. Studies from our group over the last year have provided such insights into biomedically important transporters responsible for neurotransmitter uptake, and have helped advance methodological strategies for studying their dynamics. To understand their transport mechanisms requires knowledge of the components of the protein comprising the gates that open and close on each side of the substrate binding site. For proteins whose structures have been determined at atomic resolution, these gates become immediately apparent. However, the large majority of neurotransmitter transporter proteins have not been characterized structurally. These include sodium-coupled gamma-amino butyric acid (GABA) transporters from the plasma membrane (e.g. GAT1) and proton-coupled monoamine vesicular transporters responsible for vesicle loading (e.g. VMAT2). We developed structural models of GAT1 and VMAT2, using proteins of known structure as templates in a procedure known as homology modeling. In the case of VMAT2, our model predicts residues involved with closing the cytoplasmic gate. To test this prediction, our collaborators in the Schuldiner lab (Jerusalem) modified these regions in the rat VMAT2 protein. They were able to show, using pharmacological tools (reserpine and tetrabenazine) that bind preferentially to different conformations of the protein, that these modifications alter the preferred conformation of VMAT2, in line with their predicted roles in forming the cytoplasmic gate (ref 3). A structural model of GAT1 developed in our group pointed to an unusual feature of the gate in this protein (ref 2). In particular, there is an additional amino acid inserted within a helix, which causes that helix to bulge in a structure known as a pi-helix. This bulge is specific to transporters for GABA, and not, for example for serotonin and dopamine transporters of the same protein class. Nevertheless, our collaborators in the Kanner lab (Jerusalem) could show that this bulge is key to maintaining the ratio of sodium to GABA during transport, such that if the residue is removed, the transporter appears to become leaky to cations, consistent with the idea that, in GABA transport, this region is crucial for tightly closing the pathway to the exterior of the cell. Many of the strategies that our group has adopted so far to explore transporter mechanisms have relied on static structures of the protein, often originating from X-ray crystallography, which we assume are representative of major ensembles of conformations that the protein adopts during its functional cycle. To explore the assumption of the relevant states of the cycle requires methodologies that can consider the dynamics of the protein under physiological conditions, rather than within a crystal lattice. Common strategies include double electron:electron resonance (DEER), and single-molecule fluorescence energy transfer (smFRET). However, both of these strategies require introduction of cysteine residues at multiple positions within the protein, and covalent modification of those cysteines with bulky probes, which is an arduous, and sometimes limiting strategy. An alternate approach involves using the natural tendency for exposed amide groups in the protein to exchange hydrogen atoms with those in the surrounding water, at a rate that depends on the accessibility and dynamics of each part of the protein, and that can be measured using mass spectrometry. The output of such HDX-MS measurements, however, can be an unwieldy amount of data. A few recent studies have shown that interpretation of HDX-MS data can be simplified by comparison with molecular dynamics simulations. However, to date, these studies have focused primarily on water-soluble proteins. We therefore undertook to examine the use of HDX-MS combined with molecular dynamics simulations for a test membrane protein, LeuT, a structural relative of GAT1. We showed that the simulations provide excellent agreement with the experimental data, in particular in capturing two different conformations where pathways are open on opposite sides of the membrane (ref 1). Moreover, the simulations allowed a more detailed molecular analysis of the relevant regions of the protein and their conformational dynamics and solvent accessibility. We anticipate that this strategy will therefore provide an important complement to examine the impact of substrates and inhibitors such as antidepressants on the conformational mechanism of neurotransmitter transport.